Device for bio-affinity material

ABSTRACT

Apparatus for contacting body fluids with the bio-affinity material is disclosed. The apparatus includes a container having top and bottom lock covers that clip onto the container.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.11/304,364 filed Dec. 15, 2005 now U.S. Pat. No. 7,326,182, which inturn is a continuation of application Ser. No. 10/114,314 filed Apr. 3,2002, now U.S. Pat. No. 7,014,049, which in turn is acontinuation-in-part of application Ser. No. 09/091,486 filed Jun. 19,1998, now U.S. Pat. No. 6,444,655, and a continuation-in-part ofapplication Ser. No. 09/722,241 filed Nov. 27, 2000 (now U.S. Pat. No.6,686,457) and a continuation-in-part of application PCT/SE01/00241filed Feb. 2, 2001 which are relied on and incorporated herein byreference.

INTRODUCTION AND BACKGROUND

There is a need in the art for bio-affinity materials for theextra-corporal treatment of body fluids, especially blood or bloodplasma of patients undergoing treatment for a variety of conditions,such as for example, kidney dialysis, and for a device for carrying outbio-affinity based treatments.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is described a materialcontaining a so-called matrix, to which saccharide has been bound via aspacer as is further illustrated below.

Another aspect of the invention describes a device for using the hereindescribed bio-affinity materials for their intended purpose.

The active part of the bio-affinity material according to the invention,contains at least one saccharide part which has been bound via a spacerto a matrix according to the representation: Saccharide-Spacer-Matrix.

The matrix consists in general of a polymer, plastic, or apolysaccharide, and can bind a large number of saccharide-spacer units.

The term “saccharide” as used herein symbolizes a saccharide which has abiological or other affinity to another molecule, protein, virus orcell. Saccharide can consist of a glycoprotein, aSaccharide-spacer-Matrix.

The matrix consists in general of a polymer, plastic, or apolysaccharide, and can bind a large number of saccharide-spacer units.

The term “saccharide” as used herein symbolizes a saccharide which has abiological or other affinity to another molecule, protein, virus orcell. Saccharide can consist of a glycoprotein, a neoglycoprotein, aglycopeptide or a glycosylated amino acid, a glycolipid, or a part, afragment or a modified variant thereof, or another biologically activedi- or trisaccharide or higher oligosaccharide substance.

A few non-limiting examples of biologically active saccharides, spacerand matrix which can be used according to the invention, are givenbelow.

The active part of the material consists, as a non-limiting example, ofeither:

-   -   1. Blood group A-O(CH2)nPhNH—CO—(CH₂)mNH—CH₂—CH(OH)—CH₂—O—        matrix or:    -   2. Blood group B-O(CH₂)nPhNH—CO—(CH₂)mNH—CH₂—CH(OH)—CH₂—O—        matrix

where “matrix” denotes e.g. a polymer plastic or a polysaccharide, forexample cross-linked agarose, specifically of the type Sepharose® FastFlow, where —O(CH₂)nPhNH—CO—(CH₂)mNH—CH₂—CH(OH)—CH₂— is the spacer,according to the invention, to separate the saccharide, in the aboveexamples blood group determinant A- and B-, respectively, from thematrix, where n and m, respectively, is an integer, n is for example oneof 0, 1, 2, 3 or 4, and m is for example 1, 2, 3, 4, 5, 6 or 7, andwhere the linkage between —O— and matrix is formed between —O— and, forexample, a carbon atom in the matrix.

Saccharide-spacer, for example Blood group A-O(CH₂)nPhNH—CO—(CH2)mNH—and Blood group B-O(CH2)nPhNH—CO—(CH2)mNH—, respectively, is called theligand herein. The matrix has a large number of bound molecules ofligand. Examples of bound amounts of ligand are 0.01, 0.1, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mmole perliter of matrix, or an amount of mmole which is between two of the abovegiven values per liter of matrix. Per liter matrix as used herein meansthe volume occupied by the ready-to-use matrix product.

A combination of two or more different saccharides can be used accordingto the invention, for example as non-limiting example, a combination ofBlood group A-O(CH₂)nPhNH—CO—(CH₂)mNH—, and Blood groupB-O(CH2)nPhNH—CO—(CH2)mNH—, where both ligands in this example are boundto matrix.

A feature of this invention is a device for using the bio-affinitymaterials described herein in the treatment of patients in need of suchtherapy, or for passage of blood, blood plasma, or other biologicalmaterials through the device. The bio-affinity material can, for thispurpose, be filled into a column with in- and outlet passage through thecolumn of, for example, blood plasma, or whole blood, from the patient.

The device is defined by a column filled with bio-affinity material. Ina preferred embodiment, the device is autoclavable. Different types anddifferent dimensions of column can be used. In general, the column canbe built from a cylinder, two locking covers, and between each lockingcover and the cylinder there is placed a porous membrane (or net). Eachmembrane is mounted between the locking cover and the cylinder with, forexample, a silicon ring. Each locking cover has a centrally placed holefor passage of liquid, for example, blood or plasma or other biologicalfluids, through the column. Preferred examples are given in the drawingsbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention as it relates to the device will be furtherunderstood with reference to the drawings, wherein:

FIG. 1 is a schematic diagram of the device of the present invention;

FIG. 2 is a schematic section view of a preferred example of thecylinder housing of the present invention;

FIG. 3 is a partial side view of a preferred example of the lockingmechanism used on the device of the present invention;

FIG. 4 is a schematic representation of the locking cover seen frombelow;

FIG. 5 is an elevational schematic view of the exterior cylinder housingof the present invention; and

FIG. 6 is an elevational schematic side section view of the cylinderhousing of the present invention.

DETAILED DESCRIPTION OF INVENTION

The present invention will now be described in further detail.

Turning now to the drawings, FIG. 1 shows a schematic representation ofthe apparatus or device used to carry out the present inventioninvolving contacting a body fluid such as plasma with the bio-affinitymaterials that are described herein. The device (1) includes an uprightcylinder housing (2) formed by a circumferential vertical upstandingwall (3) fitted with a top locking cover (4) and a bottom locking cover(5). The function of the covers is to close the contents of the housing(2). An outer ring (6) is optionally located at the top of housing (2)and an outer ring (7) is optionally located at the bottom of housing (2)which seals the cylinder housing (2) at the top and bottom,respectively, by securing the top and bottom locking covers. A packingring (8) and (9) is located at the bottom and top of the cylinderhousing, respectively, and fits between the respective cover (4,5) andthe housing (2). The packing ring is fitted with a retaining memberwhich can be a mesh, member or net (10) having openings of suchdimensions that it permits plasma to flow through the cylinder, butretains the bio-affinity material in said cylinder housing. The lockingcovers (4,5) are located at the top and bottom of the cylinder andfunction to secure the cylinder housing and make it tight to preventloss of contents. A cap or “propp” (11) which is preferably a screw oncap is located to cover the port or opening (12) through which thematerial as described herein is filled. Tubing (13, 14) connects thesystem for providing circulation of the fluids. The locking covers (4,5) are designed so as to form a tubular extension or projection (15,16)which enables an engaging relationship with tubing (13, 14). Theprojection extends at an angle of 90° from the plane of the lockingcover. A male luer coupling (18) is shown in mating engagement withfemale luer coupling (17) which function to connect the ends of thetubing (13, 14) together. Valves (19) and (20) are located in the tubinglines (13, 14) for controlling or stopping the circulation of the fluidin the tubing. However, in operation each liner coupling would, whenplasma treatment is desired, be connected to and used, for example,together with relevant contrifugation or plasma filtration equipment.

FIG. 2 is a cross section view showing the cylinder housing (2) which isformed of a circular wall (3). Port 12 is shown in an open state.

FIG. 3 is a cross section view and shows a portion of a locking coverwhich is used on the apparatus of the present invention to close the topand bottom of the cylinder housing (2). The locking covers consists of acircular disk-like portion (21) which has an upstanding section (22)located at angle of about 90° thereto; i.e. 90° to the plane of the disk(21). Another portion (23) of the locking cover is located on theperiphery of disk body (21) and is also bent at an angle ofapproximately 90° terminating in a cap (24) with a projection thereon tohook onto the outside edge of the cylinder (2) housing.

FIG. 4 is a bottom cross sectional view of the locking cover formed ofthe circular disk portion (21) and shows the depending portion (23) andthe caps (24) with the projection to clip onto the terminal edge of thecylinder housing.

FIG. 5 shows cylinder housing (2) in an elevational view formed of thecylinder wall (3) having the open port (12) and terminal leading edges(25, 25′) for engagement with the caps (24) of the locking cover.

FIG. 6 is a side sectional elevation view showing cylinder housing (2)and the open port (12) for filling the bio-affinity material into thecylinder housing (2).

A non-limiting example of a preferred bio-affinity material of theinvention is:

-   -   1.a. GalNAc{acute over (α)}1-3(Fuc{acute over        (α)}1-2)Galβ-O(CH₂)₂PhNH—CO—(CH₂)₅NH—CH₂—CH(OH)—CH₂—O-matrix

This type of product can be produced by reaction between for exampleGalNAc{acute over (α)}1-3(Fuc{acute over (α)}1-2)Galβ-O(CH₂)₂PhNH₂ andfor example, a carbodiimide-activated, or an NHS-activatedspacer-matrix, where matrix can be, for example, cross-linked agarose. Anon-limiting example of NHS-activated spacer matrix is NHS-activatedSepharose® 4FF, where the latter is commercially available, or othercross-linked agarose or other matrix with corresponding properties. Thereaction conditions can be chosen by the person skilled in the art anddoes not limit the scope of the invention. Other examples are productcontaining, bound in the same manner, a higher oligosaccharide than theA-trisaccharide in the above example, which contains the A-determinantterminally, for example A-determinant of type 1, 2, 3 or 4. Thetrisaccharide derivative

GalNAc{acute over (α)}1-3 (Fuc{acute over (α)}1-2)Galβ-O(CH₂)₂PhNH₂ andother saccharide derivatives mentioned in this application can beproduced with different chemical and/or biochemical methods and thisdoes not limit the scope of the invention.

Further examples of product 1 above is a product where a combination of1.a. above and one or more of mentioned blood group A variants, arebound via the same type of spacer as shown above to matrix, or via adifferent type of spacer.

A non-limiting example of a preferred variant of product 2 above whichcan be used to fill the device of this invention is:

-   -   2.b. Gal{acute over (α)}1-3(Fuc{acute over        (α)}1-2)Galβ-O(CH₂)₂PhNH—CO—(CH₂)₅NH—CH₂—CH(OH)—CH₂—O-matrix

This type of product can be produced by reaction between for exampleGal{acute over (α)}1-3(Fuc{acute over (α)}1-2)Galβ-O(CH₂)₂PhNH₂ and forexample, a carbodiimide-activated, or an NHS-activated spacer-matrix,where matrix can be, for example, cross-linked agarose. A non-limitingexample of NHS-activated spacer matrix is NHS-activated Sepharose® 4FF,where the latter is commercially available, or other cross-linkedagarose or other matrix with corresponding properties. The reactionconditions can be chosen by the skilled person in the art and does notlimit the scope of the invention. Other examples are product containing,bound in the same manner, a higher oligosaccharide, which contains theB-determinant terminally, for example B-determinant of type 1, 2, 3 or4. Further examples of product 2 are material where a combination of2.b. above and one or more of mentioned blood group B variants, arebound via the same type of spacer as above to matrix, or via a differenttype of spacer.

Instead of the —O(CH₂)₂PhNH— group in the formulas above, anothersuitable spacer or part of spacer can be used, as for example—O(CH₂)nNH— or for example N(Ac)-(CH₂)nNH— (Ac=acetyl group; n is aninteger, for example 1, 2, 3, 4, 5, 6, or 7 or higher), or anotheraliphatic compound, or another aromatic compound.

The saccharide, for example the blood group A- or B-determinant, canalso be bound, directly or indirectly, to an oligomeric substance actingas spacer, or part of spacer, as for example a mono-, di-, or higheroligosaccharide or polysaccharide, peptide, for example a peptideconsisting of amide bound glycine and glutamic acid residues, forexample Gly-(Glu-Gly)n-Glu, where n is an integer between for example 1and 20. In this manner the saccharide-spacer consists of 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more saccharide units bound to each oligomericsubstance or peptide.

The linkage between the saccharide and the peptide can for example beformed via O-glycosidically bound —O(CH₂)₂PhNH— group (see for exampleformulas 2.a. and 2.b. above), or via for example O-glycosidically bound—O(CH₂)nNH—, (where n is an integer for example 1, 2, 3, 4, 5, 6, 7 orhigher), where NH— is bound via an amide linkage (NH—CO) to the carboxylgroup on the side-chain of the Glu-residues in the peptide. —O in—O(CH₂)₂PhNH— and in —O(CH₂)nNH—, respectively, is then boundglycosidically to the saccharide.

The peptide can first have been coupled to a matrix, for example,NHS-activated matrix, such as NHS-activated Sepharose® 4FF via the□-amino-group on the peptide, and thereafter the saccharide can be boundto the peptide via —O(CH₂)₂PhNH—, or for example —O(CH₂)nNH—, to thecarboxyl group on the Glu-residues in the peptide. This linkage betweensaccharide and Glu-residues can be achieved for example, by carbodiimide(for example EDC) mediated coupling, or by for examplesuccinimide-mediated coupling. The saccharide-spacer can herewith beadded to the reaction mixture in for example a desired molar excess inrelation to the amount of moles of peptide, e.g. in a molar excess of 2,3, 4, 5, 6, 7, 8, 9 or 10 times excess or more. These and other reactionconditions are chosen by the person skilled in the art and do not limitthe scope of the invention. Non-limiting examples in this manner ofbound amount of saccharide is 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mmole of ligand per liter ofmatrix. Per liter of matrix here means the volume occupied by the readyto use matrix product.

Another example of a peptide is as above, but containing at least onelysine residue, where the ε-amino group in the lysine residue of thepeptide is used for covalent coupling to matrix, for exampleNHS-activated matrix, such as NHS-activated Sepharose® 4FF, withsubsequent coupling of, for example, saccharide-O(CH₂)₂PhNH—, or of forexample saccharide-O(CH₂)nNH—, to coupled peptide according to what hasbeen describe above. Other linkages can be used according to theinvention.

An advantage of using oligomeric ligands, as above mentioned examples ofsaccharide-peptide ligands, is that a stronger binding often can beachieved to that which is desired to be separated by the product, forexample of antibodies towards blood group determinant or of otherproteins, viruses or cells, and that therewith a more efficient productcan be obtained as compared with non-oligomeric ligand.

As a non-limiting example of one preferred variant of material accordingto the invention and as a non-limiting example of its production therecan be mentioned the coupling of about 3, 1.5 and 1 μmol, respectively,of the peptideAc-Lys-(ε-amino)-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-amidevia its ε-amino group, to 2 ml of NHS-activated matrix, such as, forexample, NHS-activated Sepharose® 4 FF, from Pharmacia-Biotech, at pH7.5 (0.2 M sodium phosphate buffer+0.5 M NaCl) under 4 h at roomtemperature followed by 0.3 M Tris-HCl, pH 8, at room temp., over night.The gel is washed with Tris-buffer and 0.1. M MES, pH 4.7, which gavePeptide-Sepharose® 4FF. Saccharide-spacer, as for exampleGalα1-3Galα-OPhNH₂, about 25 micromole, dissolved in 0.1 M MES-buffer,pH 4.7, was added to a solution of 48 mg EDC((1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide)), to which was added 2ml of Peptide-Sepharose® 4FF, the mixture was incubated under 4 h atroom temperature, after which it was washed with Tris-HCl, pH 8, 0.1 Macetate- and 0.1 M sodium phosphate buffer, respectively. The gels weretested for binding of antibodies, in the example of anti-Galα1-3Galantibodies and showed a better binding of IgM antibodies as comparedwith the same amount of saccharide-spacer-Sepharose® 4FF, obtained bycoupling of the corresponding amount of Galα1-3Galα-OPhNH—, but withoutpeptide, directly to NHS-activated Sepharose® 4FF. Other saccharidederivatives than Galα1-3Galα-OPhNH₂ can be used according to theinvention, such as for example GalNAcα1-3(Fucα1-2)Galβ-O(CH₂)₂PhNH₂ orGalα1-3(Fucα1-2)Galβ-O(CH₂)₂PhNH₂.

The saccharide-peptide conjugate can for example first be prepared byusing the ε-BOC-derivative (BOC=tert-butyloxycarbonyl group), situatedon the ε-amino group of the lysine residue of the peptide in the abovementioned example. The saccharide-spacer-peptide conjugate is then firstformed by for example EDC-mediated reaction between amino groups on thesaccharide-spacer and the carboxyl groups on the peptide, the resultingconjugate can be purified by, for example, Sephadex chromatography, theBOC group can be eliminated, by for example, trifluoroacetic acidreaction according to standard conditions for peptide chemistry, and theconjugate can be coupled, for example, in the same manner as describeabove via the ε-amino group of the lysine residue to the matrix, forexample, a carbodiimide-activated, or an NHS-activated spacer-matrix,where matrix can be, for example, cross-linkagarose. A non-limitingexample of NHS-activated spacer-matrix is NHS-activated Sepharose® 4FF,where the latter is commercially available, or other cross-linkedagarose or other matrix with corresponding properties.

As another non-limiting example of peptide there can be mentionedpeptide consisting of amide bound Gly and Lys units, for exampleGly-(Lys-Gly)n-Gly, where n is an integer between for example 1 and 20.In this case for example the peptide can be bound to the saccharide viaamino groups on the peptide, a N-glycosidic linkage is formed betweenthe reducing end on the saccharide and the ε-amino group on the lysineresidue(s), and the saccharide-peptide can be coupled to the matrix via,for example, either the remaining amino group(s) on the peptide to forexample NHS-activated Sepharose® 4FF as described above, or via, forexample, the terminal COO-group on the peptide and amino groups on aminogroup containing matrix, for example aminohexyl-Sepharose® (by forexample carbodiimide or succinimide coupling according to examples givenabove). The N-glycosidic linkage can be stabilized by acetylation understandard conditions, for example before coupling to the matrix, e.g.NHS-activated Sepharose® 4 FF. In the same manner as for theGly-Glu-peptide above also an aliphatic or aromatic spacer can be usedto bind the saccharide to the lysine residues of the peptide, but inthis case is, for example, glycosidically bound groups of the type—O(CH₂)₂PhCOO—₂ or for example —O(CH₂)nCOO—, are used for carbodiimide-or succinimide-mediated coupling between saccharide and lysine residuesin the peptide.

The coupling to the peptide can also be carried out by first couplingthe saccharide part to the amino acid and thereafter form the peptidelinkages.

Further examples of ligands according to the invention, is to use aprotein or a polysaccharide as spacer, or part of the spacer, betweensaccharide and matrix. Here for example a protein, such as serumalbumin, or a polysaccharide, such as dextran, is used. The saccharidecan then first be coupled to the protein, or to the polysaccharide,which then is coupled to the matrix. The same type of chemistry asexemplified above can, as non-limiting examples, be used to achieve thelinkages between saccharide, protein, or polysaccharide, and matrix.This does not limit the scope of the invention, and the conditions arechosen by the expert.

To use a peptide, protein or polysaccharide according to what haveexemplified above, can in some cases be an advantage to increase theability of the material to bind protein, and thereby increase theefficiency of the product according to the invention.

As another example of matrix there can be mentioned the filters whichare used for plasma separation. These can be chemically modified withstandard technique and be used for coupling of oligomeric ligand or ofnon-oligomeric ligand mentioned in this description. In this mannerproduct is achieved which can be used for specific removal of proteinsin connection with blood plasma separation, for example antibodiesdirected towards Galα1-3Gal and other so called xeno antigens inconnection with xenotransplantation.

In a variant of the invention, the product in addition contains a Trisstructure according to the following non-limiting example:(HOCH₂)₃C—NH—CO—(CH₂)₅NH—CH₂—CH(OH)—CH₂—O-matrix where (HOCH₂)₃C—NH— isa so called Tris-group. This product can be made by reaction betweenTris-HCl and for example, NHS-activated spacer-matrix, where matrix canbe cross-linked agarose. A non-limiting example of NHS-activatedspacer-matrix is NHS-activated Sepharose® 4FF.

In the production of the product according to the invention there can beused, for example, commercially available activated matrix, for exampleso called NHS-activated Sepharose® 4 Fast Flow (NHS- is an abbreviationof N-hydroxysuccinimide; this variant of agarose is relatively stronglycross-linked, commercially available), which is present in the form ofpractically spherical particles. The particle size is chosen in, forexample, the interval 45-165 μm. This activated matrix can be used forcovalent binding of for example, Blood group A-O(CH₂)_(n)PhNH₂—, to giveproduct 1.a. above, and of Blood group B-O(CH₂)_(n)PhNH₂, which giveproduct 2.b. above, respectively, at, as non-limiting and typicalexamples, pH 7.5 or pH 8.0, in buffer, for example 0.1 M sodiumphosphate as non-limiting example, under for example 1, or 2 hours orfor 20 hours, and in the example at room temperature. After thereaction, the material is washed for example on a glass filter or underother conditions, for example sterile conditions, with, for example,buffer and is subsequently treated with for example Tris-HCl buffer toreact any remaining reactive groups. The person skilled in the art willbe able to choose the conditions for the reactions and this does notlimit the scope of the invention. See also the above given example inconnection with the preparation of Galα1-3Gal-Peptide-Sepharose® 4FF.

In the production of the product according to the invention there can,as another example, be used the so-called epoxy-activated Sepharose® 4Fast Flow, to which is covalently bound, for example

-   -   Blood group A-O(CH₂)_(n)PhNH—CO—(CH₂)_(m)NH—, or to which is        covalently bound    -   Blood group B-O(CH₂)_(n)PhNH—CO—(CH₂)_(m)NH—,        where n or m are specified above as are Blood group A and Blood        group B, respectively.

As has been mentioned above, a combination of ligands can also becovalently bound to the matrix.

The products can be used, for example, for extra-corporal removal ofblood group A- and blood group B-antibodies, respectively, e.g. forpurification of blood, or for example, before a transplantation, forexample over the blood group barrier. The product can be used in generalfor different types of transplantation as a part of the treatment of therecipient before and during, and eventually after the transplantation.Lowering of anti-A and/or anti-B antibody titers have been shown inseveral studies to facilitate cross-ABO transplantation. Thus, anti-Aantibodies of a blood group O, or a blood group B patient are lowered orremoved before transplantation of a blood group A organ, byextra-corporal treatment as described above of the blood or blood plasmaof the patient, with the device according to the invention containingblood group A. Moreover, anti-B antibodies of a blood group O, or ablood group A patient can be lowered or removed before transplantationof a blood group B organ, by extra-corporal treatment as described aboveof the blood or blood plasma of the patient, with the device accordingto the invention containing blood group B. This to be able to solve theproblem of blood group incompatibility between donor and recipient. Thebio-affinity material can for this purpose be filled into a columnhousing with in- and outlet for passage through the column of forexample blood plasma, or whole blood, from the patient who shall betransplanted or who is undergoing a transplantation procedure. The useof the product is therefore not restricted to for example, blood groupincompatible transplantation, but can also be used, for example, forblood group compatible transplantation, to minimize problems inconnection with donor and recipient of the same blood group, but ofdifferent blood group subgroups, for example A1, A2 etc.

Other non-limiting examples of saccharide according to the specificexamples 1 or 2 above, are structures where the saccharide part consistsof Galα1-3Galα-, Galα1-3Galβ-, Galα1-3Galβ1-4Glcβ-,Galα1-3Galβ1-4GlcNAcβ-, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-, or ofoligomeric ligands, such as for example (Galα1-3Galα-)n-,(Galα1-3Galβ-)n-, (Galα1-3Galβ1-4Glcβ-)n-, (Galα1-3Galβ1-4GlcNAcβ-)n-,(Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-)n-, or (Galα1-3Galα-spacer)n-,(Galα1-3Galβ-spacer)n-, (Galα1-3Galβ1-4Glcβ-spacer)n-,(Galα1-3Galβ1-4GlcNAcβ-spacer)n-,(Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-spacer)n-, where n is an integerlarger than 1. As mentioned above for other saccharides, differentspacers can be used in connection with above mentioned saccharides.Non-limiting examples of the spacer have been given above, for exampleof non-oligomeric, oligomeric, and of polymeric type, respectively.

These structures can be of interest to be used in for example a columnor in a plasmafilter, for example before and after xenotransplantationto reduce so called xeno-antibodies from the patient's blood (wholeblood column) or plasma.

Other carbohydrate structures active towards other antibodies, forexample antibodies against cancer-antigens, for example prostate-,breast-, intestine-, or skin cancer, can be used to form productaccording to the invention. This can be of interest for example toisolate antibodies from blood or plasma against said antigen and afterelution from the product, be used for treatment of said cancer diseases,or to produce reagents, or for example, to remove an excess of antibodyderivatives from blood or plasma in immunotherapy of cancer. Othercarbohydrate structures specific for e.g. toxins, virus and/or bacteria,can also be used to form product according to the invention. Theseproducts can be used to, for example, purify or eliminate virus and/orbacteria from e.g. whole blood or plasma or from other materials, forexample food products or from water.

A combination of two or more different ligands (Saccharide-spacer) boundto matrix, can be used in the product according to the invention. Thesaccharides can then be different, and/or the spacer can be different.

The product according to the invention, allows for example a combinationof high flow rate (for example in the interval 20-60 ml/min), minimaldrop in pressure over the column, and a good binding capacity also ofmolecularly larger proteins, for example antibodies, such as IgG andIgM. As a non-limiting example there can be mentioned a single passageof more than one liter blood group B plasma with a flow rate of about 40ml/minute through a column with about 3 micromol of blood group Atrisaccharide per ml Sepharose® 4 FF, and with a total product volume of62 ml, and an average particle size of 90 μm, practically eliminated allantibodies reactive against blood group A. Similar result was obtainedwith blood group B product. The products were built according to 1.a.and 2.b. above from A- and B-trisaccharide-spacer and NHS-activatedSepharose® 4 Fast Flow.

One or more of the devices according to the invention can be used inparallel or one device coupled after the other. Thus, for example, onedevice containing bio-affinity material with blood group A and onedevice containing bio-affinity material with blood group B can be usedin parallel or in sequence to remove both anti-A and anti-B antibodiesfrom blood or blood plasma, thus creating, for example, universal plasmawhich can be given to patients irrespective of blood group of thepatient. The column volume is chosen for the purpose and can be forexample of a size of, for example, 10 ml, 20 ml, 40 ml, 60 ml up to asize of for example 500 ml or higher depending on which volumes aredesired to process. The column volume can be for example of a valuebetween the given values. Different types of column housing of differentdimensions can be used. The bio-affinity material according to theinvention functions, as non-limiting example, in the type of columnhousing with the dimensions used for the product ImmunoSorba®, (whichhas protein A as ligand bound to matrix), which has an inner volumebetween the porous membranes of about 62 ml (that is allows filling of62 ml material according to the invention).

When using products according to the invention for treatment of plasma,in general there can be used membranes which have a lower porosity andmatrix particles which have lower particle size as compared with thecase when the product is applied for treatment of whole blood. Thus, forexample, in the case of the treatment of plasma, membrane with porosityof for example 30 micrometer, thus retaining particles of diameterlarger than 30 μm, or membranes with a porosity in the range 20 to 40micrometer, and particle size of matrix of for example 90 micrometer, ormatrix of for example particle size in the range 40-200 micrometer, canbe used. When using products according to the invention for treatment ofwhole blood, membranes with porosity of for example 30 micrometer or 70micrometer, or membrane with a porosity in the range 20 to 100micrometer, can be used, and the particle size of the matrix can be forexample 150 micrometer, or the matrix particle size can be for examplein the interval 100-250 micrometer. The porosity is chosen by the personskilled in the art and does not limit the scope of the invention.

The filling of the bio-affinity material according to the invention intothe column can be done using different principal methods. According tothe invention, the bio-affinity material can for example either beautoclaved first and thereafter be filled aseptically in the column, orthe bio-affinity material can, as an example of another preferred formof the invention, first be filled in the column and thereafter thecolumn with the bio-affinity material is autoclaved.

Non-limiting example of autoclaving is treatment in an autoclave of forexample counter-pressure type, which involves treatment under at least20 minutes at 121° C. or higher and with for example water steam. Otherconditions can be chosen by the person skilled in the art from what issuitable, e.g. sterility and stability of the product. As an examplethere can be mentioned that the Saccharide-spacer-Matrix according toexamples 1.a. and 2.b., obtained via coupling of the respective ligandto NHS-activated Sepharose® 4FF, exhibits the same properties afterautoclaving as before autoclaving concerning tested parameters such asantibody binding properties and other properties.

The column either completely, or partially, filled with materialaccording to the invention, can for example be constructed in materialswhich allows for autoclaving (biocompatible plastic materials which canbe autoclaved are commercially available, e.g. column housing andlocking covers are made of special polycarbonate, tubings of PVC orsilicon material and rings of silicon) and/or for example to allowaseptic packing of material according to the invention. Column housingsexists on the market for extracorporal blood treatment, e.g.Immunosorba. This allows for aseptic filling, but not for autoclaving.

Non-limiting example of autoclavable column housings as illustrated inone embodiment thereof in FIG. 1 include a column housing (2) built fromautoclavable materials. The column housing typically has two lockingcovers (4,5). Between each locking cover and cylinder and beforeplacement of the locking covers (4, 5) onto the cylinder, there isplaced a porous membrane (9, 10) (that is two membranes and rings foreach column), which allows for passage of plasma or whole blood but notfor passage of the bio-affinity material according to invention. Eachmembrane is mounted between the locking cover and the cylinder with forexample a silicon ring with a fitting groove of the same or about thesame diameter as the cylinder. Every silicon ring has for example agrove which allows for fitting the circular membrane in the groove inthe silicon ring. The membrane is mounted in the silicon ring and isplaced between the locking cover and the ending of the cylinder housing,after which each locking cover is fitted onto the cylinder. The siliconring with the membrane therewith is enclosed between the locking coverand the cylinder ending. The same procedure is carried out for the otherending of the cylinder. Each locking cover has a centrally placed holewith a projection (15, 16) which allows for connecting a bio-compatibleand autoclavable set of tubings (13, 14) equipped with connections (17,18) of e.g. the luer type for connection of other equipment used inextracorporal treatment.

It is preferred that the locking covers and the cylinder are connectedwith a clip mechanism where for example the locking covers are equippedwith one or more clips (for example in each one of the locks there are2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more separate clips whichare situated beside each other with a defined interspace), and thecylinder has on its outer side one or more protruding edge(s) placed ata distance, e.g. of 2, 3, 4, 5, or 6 mm from the top and from thebottom, respectively, of the cylinder. The clips can be protruding downfrom the locking covers and can for example be homogeneous, or each onebe equipped with a cavity to allow for a larger flexibility withoutbeing broken. In this manner the silicon ring with the membraneaccording to the above can be placed between the locking cover and therespective cylinder ending, and the locking cover is thereafter pressedonto the cylinder, whereupon the clips are pressed under the protrudingedges on the cylinder and stays there, and the silicon ring with theporous membrane is consequently sealed between the locking cover and thecylinder housing.

Alternatively the locking covers and the top and bottom edges ofcylinder housing (2) can be equipped with matching screw threads.

In order to fill the so mounted column housing with material accordingto the invention, the cylinder part can be equipped with a circularopening (12) with a protruding part, which has threads, on the outerwall of the cylinder to allow for connection of tubing used for fillingof the material. After filling of the material into the column housing,a bio-compatible plug (11) with threads which matches the threads of theprotruding part on the cylinder, is mounted. In the center of the plugis a protruding tap which fits into the circular hole of the cylinderand which has a length which corresponds to the height of the protrudingpart. In this manner an almost flat surface is achieved inside thecylinder at the circular opening.

For the autoclaving of a column filled with bio-affinity materialaccording to the invention and connected with a set of tubings, an outerring of e.g. PVC material, and with an inner diameter which correspondswith the outer diameter of the column, can be mounted around the eachlocking cover before the autoclaving. This can be done in order tominimize any deformation of the cylinder under the autoclaving process.This principle has been successfully used under autoclaving of columnfilled with material according to the above.

All mentioned components of the column house in a preferred exampleaccording to the invention with autoclavable column house, areautoclavable and biocompatible. Locking cover, membrane, cylinder, plug,and tubing with luer coupling can be made of biocompatible plasticmaterial. For example, in the preferred embodiments, the cylinder andlocking covers can be fabricated from polycarbonate, the ring of siliconrubber, the membrane net of polyester, tubing and luer couplings of PVCand the plug of acrylobuta dienestyrene which are all bio-compatible.

Column housing completely or partially filled with bio-affinity materialaccording to the invention and equipped with above mentioned closedtubing set and plug can be autoclaved. According to the invention thisfacilitates the achievement of sterility of the flow path of theproducts. With earlier methods sterile (aseptic) fillings have beencarried out, but are difficult to achieve.

The invention claimed is:
 1. An autoclavable apparatus comprising: acylindrical container defined by a circumferential wall, having a topend and a bottom end, each of said top end and said bottom end having aprotruding edge outward from the wall, a port defining an opening insaid circumferential wall, said port protruding from the circumferentialwall in an outward direction, a top lock cover in the form of a circulardisk which has a plurality of downward projections of sufficientflexibility to clip on to and under the protruding edge of saidcylindrical container, said top lock cover being attachable to the topend of said container, said top lock cover having a central opening, abottom lock cover in the form of a circular disk which has a pluralityof upward projections of sufficient flexibility to clip onto and overthe protruding edge of said cylindrical container, said bottom lockcover being attachable to the bottom end of said container with saidbottom lock cover having a central opening, said downward projectionsbeing of sufficient flexibility to engage with the protruding edge ofsaid container and to permit said top lock cover to be pressed onto thecontainer, optionally an outer ring located outside and adjacent to thetop and bottom of the cylindrical housing to engage with and secure thetop lock cover and bottom lock cover, respectively, a cover for saidport, a first packing ring positioned between the upper end of saidcontainer and said top lock cover, a second packing ring positionedbetween the bottom end of said container and said bottom lock cover,said first and second packing ring each being fitted with a membranelocated adjacent each of the top end and the bottom end of saidcontainer, which is filled with a bio-affinity material which is (a) anA-trisaccharide represented by the structure: GalNAcα 1-3 (Fuc α 1-2)Gal β —Z—CO—(CH₂)₅NH—CH₂—CH(OH)—CH₂—O— matrix, or (b) a B-trisacchariderepresented by the structure: Gal α 1-3 (Fuc α 1-2) Gal β—Z—CO—(CH₂)₅NH—CH₂—CH(OH)—CH₂—O— matrix, wherein the matrix iscross-linked agarose, and Z is a member selected from the groupconsisting of —(CH₂)₂PhNH—, —O(CH₂)_(n)NH— and —N(Ac)-(CH₂)_(n)NH; wheren=1-7 and Ac is acyl.
 2. The apparatus according to claim 1, whereineach said membrane is porous for passage of liquid but which can retaina biologically active material.
 3. The apparatus according to claim 1,formed of biocompatible materials.
 4. The apparatus according to claim1, where each membrane has a porosity of in the range of 20 to 100micrometer.
 5. The apparatus according to claim 1, wherein the membranehas a porosity capable of retaining particles with a mean particle sizeof 40-250 micrometer.
 6. The apparatus according to claim 1, where saidcontainer has an inner volume in the range of 10 ml to of 500 ml.
 7. Theapparatus according to claim 1, further comprising a tubing connected toeach of the lock covers and two luer couplings for connecting saidtubing.
 8. The apparatus according to claim 7, where the tubing and luercouplings are biocompatible.
 9. The apparatus according to claim 1,where the plurality of downward and upward projections are clips, whichare situated beside each other with a defined interspace, the containerhaving on its outer side a protruding edge near the top and the bottom,respectively, of the container, said clips protruding down from the toplock cover and up from the bottom lock cover and being of sufficientflexibility to engage with the protruding edge of the container, and topermit both top and bottom lock covers to be pressed on the container,whereupon the clips are pressed under the protruding edges on each lockcover.
 10. The apparatus according to claim 1, wherein the outer ring atthe top and bottom is of plastic and have an inner diameter whichcorresponds with the outer diameter of each lock cover.
 11. Theapparatus according to claim 1, where the column housing and lock coversare made of biocompatible polycarbonate and tubings are PVC or siliconpolymer.
 12. The apparatus according to claim 1, which additionallycontains at least one higher oligosaccharide of A-determinant type 1, 2,3 or
 4. 13. The apparatus according to claim 1, which additionallycontains at least one higher oligosaccharide of B-determinant type 1, 2,3 or
 4. 14. The apparatus according to claim 1, wherein Z is—(CH₂)₂PhNH—.